Regulatory T lymphocytes (Tregs) have been implicated in preserving immunologic homeostasis and their absence may lead to autoimmunity. During the previous funding period of this competitive renewal application, we have uncovered and characterized a novel population of CD8 Tregs in humans. These CD8 Tregs are inducible through IL-15 combined with low-affinity T cell receptor stimulation; they express a unique phenotype; CD8+CD45RA+CCR7+ and function by disrupting membrane-proximal signaling events in CD4 T cells. Specifically, CD8+CCR7+ Tregs prevent phosphorylation of the adaptor molecule Zap70 in adjacent CD4 T cells undergoing activation. In patients with rheumatoid arthritis (RA) and in elderly individuals induction of these CD8 Tregs is markedly impaired. Generation of CD8+CCR7+ Tregs is enhanced in the presence of the mTOR inhibitor Rapamycin. The current application is designed to understand, on a molecular level, how CD8+CCR7+ are induced; why induction is hampered in patients with rheumatoid arthritis and through which pathways adoptively transferred CD8+CCR7+ Tregs suppress the inflammatory activity of rheumatoid synovitis. Finally, we will explore how CD8+CCR7+ Tregs communicate with adjacent CD4 T cells and seek to identify receptor-ligand pairs mediating the negative signal. Specific Aim 1 is designed to examine the transcriptional regulation of FOXP3 in successful and failed induction of CD8+CCR7+ Tregs and to investigate how Rapamycin-modulated signaling pathways can be exploited to optimize generation. Specific Aim 2 is devoted to define the precursor cells giving rise to CD8+CCR7+ Tregs and to mechanistically link the defective DNA repair machinery in RA with the failure to produce functional CD8 Tregs. Specific Aim 3 is focused on confirming and extending studies of the in vivo function of CD8+CCR7+ Tregs in suppressing rheumatoid synovitis; relying on a human synovium-SCID chimera model. Specific Aim 4 is dedicated to unravel the molecular mechanism through which CD8 Tregs prohibit CD4 T cells from proceeding with TCR-initiated activation. Cell imaging studies will decipher the membrane architecture of the CD4-CD8 T cell interaction platform and a whole genome siRNA screen will serve as an unbiased discovery tool to identify receptor-ligand pairs transmitting the negative signal.